Polypeptide substrate for the detection of von Williebrand factor cleaving protease ADAMTS13

ABSTRACT

In a first aspect, there is provided an isolated polypeptide substrate for a disintegrin-like and metallopeptidase with thrombospondin type-1 motif, 13 (ADAMTS13) that is from 45 to 70 amino acids in length and has an amino acid sequence that is substantially similar to part of the von Willebrand factor A2 domain sequence set forth in SEQ ID NO: 2, with one or more of the following modifications: (i) the amino acid corresponding to position 1599 of SEQ ID NO: 2 is mutated from Q to K; (ii) the amino acid corresponding to position 1610 of SEQ ID NO: 2 is mutated from N to C; and (iii) the amino acids corresponding to Q1624 to R1641 of SEQ ID NO: 2 are deleted. In another aspect, there is provided an ADAMTS13 polypeptide substrate that is from 50 to 75 amino acids in length and has an amino acid sequence that is substantially similar to part of the von Willebrand factor A2 domain sequence set forth in SEQ ID NO: 2, with one or more of the following modifications: (i) the amino acid corresponding to position 1599 of SEQ ID NO: 2 is mutated from Q to K; (ii) the amino acid corresponding to position 1610 of SEQ ID NO: 2 is mutated from N to C; (iii) the amino acid corresponding to position 1629 of SEQ ID NO: 2 is mutated from G to E; and (iv) the amino acids corresponding to G1631 to R1641 of SEQ ID NO: 2 are deleted.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/357,988, filed May 13, 2014, which is a National Stage application ofPCT International Application No. PCT/US2012/064526, filed Nov. 9, 2012,which claims the benefit of priority to U.S. Provisional Application No.61/558,927, filed on Nov. 11, 2011, the entire contents of which arehereby incorporated by reference.

BACKGROUND

von Willebrand factor (VWF) is a large multimeric plasma glycoproteincrucial in the maintenance of hemostasis by functioning as both anantihemophilic factor carrier and a platelet-vessel wall mediator in theblood coagulation system, mainly by mediating tethering and adhesion ofcirculating platelets at sites of vascular injury. Mutations in thisgene or deficiencies in this protein result in von Willebrand's disease(VWD).

VWF is expressed by endothelial cells and megakaryocytes. It issynthesized as 250-kDa monomers, which undergo intracellular processing,glycosylation, multimerization and propeptide removal that leads toformation of mature VWF multimers.

VWF multimeric size is modulated by the plasma metallopeptidase ADAMTS13(a disintegrin and metallopeptidase with thrombospondin type 1 motif,member 13, a “cleaving protease”), which cleaves at a single site in theVWF A2 domain (AA1498-1665; UniProtKB/Swiss-Pro database; Accession:P04275. SEQ ID NO: 2) between Y1605 and M1606.

ADAMTS13 is a protease that is activated in the presence of barium andother metal ions. ADAMTS13 has been demonstrated to degrade full-lengthmultimeric vWF into multimers of smaller size and into lower molecularweight polypeptides or peptides. For this reason, the ADAMTS13 proteasehas been termed vWF-cleaving protease or the “ATS protease”. Theactivity of the protease has been demonstrated to be reduced in patientswith Thrombotic Thrombocytopenia Purpura (TTP).

Severe deficiency of the protease has been described in patients withchronic relapsing TTP, a deficiency that may be inherited or acquired asa result of an autoimmune mechanism.

In the past, assays for the presence or absence of ADAMTS13 utilized acumbersome technique in which plasma from a patient is incubated withexogenous multimeric vWF in the presence of barium chloride on thesurface of a membrane floating on a buffer containing 1.5 molar urea.More recently an alternative method has been developed by Kokame et al.(Kokame, K., Y. Nobe, Y. Kokubo, A. Okayama, and T. Miyata. 2005.FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br. J.Haematol. 129:93-100. See also Wu J J, Fujikawa K, McMullen B A, Chung DW. Characterization of a core binding site for ADAMTS13 in the A2 domainof von Willebrand factor. Proc Natl Acad Sci USA. 2006; 103: 18470-4.).Kokame's method utilizes a polypeptide substrate for ADAMTS13 activity,wherein the substrate is 73 amino acid residues in the A2 domain of VWF,called VWF73. FRETS-VWF73 is within this domain and the 73-amino-acidpolypeptide sequence corresponds to the region from D1596 to R1668 ofVWF (see SEQ ID NO: 6 herein), Q1599 and N1610 when substituted withA2pr(Nma) and A2pr(Dnp) respectively. Several assays have been developedusing SEQ ID NO: 6. VWF73-based ADAMTS13 assays have the potential tocontribute to improved clinical treatments.

However, the de novo synthesis of SEQ ID NO: 6 is difficult and theFRETS-VWF73 substrate works near the UV spectrum. The signal that isgenerated therefore suffers from heavy contribution of autofluorescencewhich can be exacerbated by the fact that the assay is homogeneous, i.e.is performed in a single step without washing away the plasma, one ofthe major contributors to the autofluorescence noise. Because of itssusceptibility to autofluorescence, an assay based on the FRETS-VWF73substrate is very sensitive to dust microparticles, potentiallyresulting in poor replicates and aberrant results. Furthermore,FRETS-VWF73 substrate assays typically result in a non-linearcalibration curve which can result in low accuracy below 10% of ADAMTS13activity. This is problematic since the resolution of ADAMTS13 activityat between 0-10% is important to clinicians to confirm the diagnosis ofTTP and to monitor and fine tune the therapeutic intervention (such asplasma exchange). Further, ADAMTS13 activity assays using a SEQ ID NO: 6polypeptide suffer from poor sensitivity.

As a result, there is a need in the art for an improved ADAMTS13polypeptide substrate. The present invention seeks to address this need.

SUMMARY

In a first aspect, there is provided an isolated polypeptide substratefor a disintegrin-like and metallopeptidase with thrombospondin type-1motif, 13 (ADAMTS13) that is from 45 to 70 amino acids in length and hasan amino acid sequence that is substantially similar to part of the vonWillebrand factor A2 domain sequence set forth in SEQ ID NO: 2, with oneor more of the following modifications: (i) the amino acid correspondingto position 1599 of SEQ ID NO: 2 is mutated from Q to K; (ii) the aminoacid corresponding to position 1610 of SEQ ID NO: 2 is mutated from N toC; and (iii) the amino acids corresponding to Q1624 to R1641 of SEQ IDNO: 2 are deleted.

In a second aspect, there is provided an ADAMTS13 polypeptide substratethat is from 50 to 75 amino acids in length and has an amino acidsequence that is substantially similar to part of the von Willebrandfactor A2 domain sequence set forth in SEQ ID NO: 2, with one or more ofthe following modifications: (i) the amino acid corresponding toposition 1599 of SEQ ID NO: 2 is mutated from Q to K; (ii) the aminoacid corresponding to position 1610 of SEQ ID NO: 2 is mutated from N toC; (iii) the amino acid corresponding to position 1629 of SEQ ID NO: 2is mutated from G to E; and (iv) the amino acids corresponding to G1631to R1641 of SEQ ID NO: 2 are deleted.

Suitably, the amino acid at the N-terminus of said polypeptide substratecorresponds to D1596 of SEQ ID NO: 2.

Suitably, the amino acid at the C-terminus of said polypeptide substratecorresponds to R1668 of SEQ ID NO: 2.

Suitably, the polypeptide is a synthetic polypeptide that comprises adetectable label.

Suitably, the detectable label is a fluorophore and a quencher.

Suitably, the attachment site for the fluorophore is at the amino acidcorresponding to position 1610 of SEQ ID NO: 2 and/or wherein theattachment site for the quencher is at the amino acid corresponding toposition 1599 of SEQ ID NO: 2 or wherein attachment site for thequencher is at the amino acid corresponding to position 1610 of SEQ IDNO: 2 and/or wherein the attachment site for the fluorophore is at theamino acid corresponding to position 1599 of SEQ ID NO: 2.

Suitably, the ADAMTS13 polypeptide substrate comprises, consists orconsists essentially of the sequence set forth in SEQ ID NO: 7.

Suitably, the ADAMTS13 polypeptide substrate comprises, consists orconsists essentially of the sequence set forth in SEQ ID NO: 1.

Suitably, the ADAMTS13 polypeptide substrate is lyophilized.

In a further aspect, there is provided a method for cleaving theADAMTS13 polypeptide substrate, comprising contacting said ADAMTS13polypeptide substrate with an ADAMTS13 protease.

In a further aspect, there is provided a method for measuring ADAMTS13activity in a sample comprising the use of the ADAMTS13 polypeptidesubstrate.

Suitably, the method comprises the steps of: (a) providing a samplecomprising, or suspected of comprising, an ADAMTS13; (b) contacting saidsample with the ADAMTS13 polypeptide substrate; and (c) determining thefragmentation of the ADAMTS13 polypeptide substrate, wherein thefragmentation of the ADAMTS13 polypeptide substrate is optionallycompared to one or more controls and/or calibrators in order to arriveat a measurement of ADAMTS13 activity.

Suitably, the cleavage of the ADAMTS13 polypeptide substrate is measuredby monitoring the change in fluorescence.

Suitably, the sample at step (a) is a plasma sample or is derived from aplasma sample.

Suitably, the ADAMTS13 polypeptide substrate is in solution duringcontacting step (b). Suitably, the ADAMTS13 polypeptide substrate is insolution when cleaved by a protease. Suitably, the ADAMTS13 polypeptidesubstrate is in solution when cleaved by an ADAMTS13 protease.

Suitably, the ADAMTS13 polypeptide substrate is attached to a solidsupport during contacting step (b). Suitably, the ADAMTS13 polypeptidesubstrate is attached to a solid support when cleaved by a protease.Suitably, the ADAMTS13 polypeptide substrate is attached to a solidsupport when cleaved by an ADAMTS13 protease. Suitably, the ADAMTS13polypeptide substrate is attached to a well during contacting step (b).Suitably, the ADAMTS13 polypeptide substrate is attached to two or morewells of a microwell strip during contacting step (b). Suitably, theADAMTS13 polypeptide substrate is attached to a bead during contactingstep (b).

Suitably, step (d) is a quantitative determination of the fragmentationof the ADAMTS13 polypeptide substrate.

In a further aspect, there is provided a kit for in vitro testing ofADAMTS13 activity in a subject, comprising the ADAMTS13 polypeptidesubstrate, one or more calibrators containing a known concentration ofADAMTS13 activity and/or one or more positive controls for ADAMTS13activity optionally together with a specimen diluent and/or a substratebuffer.

In a further aspect, there is provided the use of the ADAMTS13polypeptide substrate for measuring the activity of ADAMTS13 protease ina sample.

The ADAMTS13 polypeptide substrates that are described herein have anumber of advantages.

By way of example, the polypeptide substrate can be reliablysynthesised. When the polypeptide substrate is synthesized by chemicalsynthesis it can be produced at lower cost as compared to recombinantsynthesis and 73-mer synthesis.

By way of further example, a linear calibration curve can be achievedalong with higher resolution, sensitivity and precision as compared tothe existing ADAMTS13 activity-based assays.

By way of further example, reduced signal-to-noise ratio in the ADAMTS13assay can be obtained.

By way of further example, faster reaction time (15 minutes or lessreaction time vs. the 30 minutes required by the FRETS-VWF73-basedassay) in the ADAMTS13 assay can be obtained.

By way of further example, when detectable labels are used, excitationand emission occurs at the most widely used wavelengths which makesdetection simpler.

By way of further example, a higher dynamic range of the assay can beachieved resulting in the ability to precisely determine ADAMTS13 in therange of about 0-20% activity, a range that cannot be efficientlyresolved in the existing activity-based assay. Thus, improvements in thedifferential diagnosis of TTP from other disorders including hemolyticuremic syndrome (HUS), which present similar clinical symptoms, can beachieved. Improvements in the prognostic management of TTP can also beachieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein sequence of a portion of VWF (AA1495-1668 ofSEQ ID NO: 2) encompassing the A2 domain (AA1498-1665).

FIG. 2 shows the amino acid sequence of SEQ ID NO: 1.

FIG. 3 displays a series of calibration curves obtained with the priorart FRETS-VWF73.

FIG. 4 displays a series of calibration curves obtained with Applicantssynthetic 62 (sixty two) amino acids in length polypeptide sequencedesignated as “GTI_FRET4” SEQ ID NO: 1.

FIG. 5 displays a series of calibration curves obtained with Applicantssynthetic 55 (fifty five) amino acids in length polypeptide sequencedesignated as “GTI_FRETS” SEQ ID NO: 7 Showing a change in fluorescencewith time.

DETAILED DESCRIPTION Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention. The technical terms and expressions used within the scopeof this application are generally to be given the meaning commonlyapplied to them in the art. All of the following term definitions applyto the complete content of this application. The word “comprising” doesnot exclude other elements or steps, and the indefinite article “a” or“an” does not exclude a plurality. The terms “essentially”, “about”,“approximately” and the like in the context of a given numerate value orrange refers to a value or range that is within 20%, within 10%, orwithin 5% of the given value or range. Due to the imprecision ofstandard analytical methods, molecular weights and lengths of polymersare understood to be approximate values. When such a value is expressedas “about” X or “approximately” X, the stated value of X will beunderstood to be accurate to +10%.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, amplification products, fragments generated byany of ligation, scission, endonuclease activity, and exonucleaseactivity, genomic DNA, recombinant vectors and chemically synthesizedmolecules. Nucleic acid molecules can be composed of monomers that arenaturally-occurring nucleotides, or analogs of naturally-occurringnucleotides (e.g., alpha-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Nucleic acids can be eithersingle stranded or double stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic nucleic acid of an organism. For example,a nucleic acid molecule that has been separated from the genomic nucleicacid of a cell is an isolated nucleic acid molecule. Another example ofan isolated nucleic acid molecule is a chemically-synthesized nucleicacid molecule that is not integrated in the genome of an organism. Anucleic acid molecule that has been isolated from a particular speciesis smaller than the complete nucleic acid molecule of a chromosome fromthat species.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups, fluorescent detection moieties and/or linkers.These non-peptidic components may be added to a protein by the cell inwhich the protein is produced, and will vary with the type of cell.Proteins are defined herein in terms of their amino acid backbonestructures; non-peptidic components are generally not specified whengenerally referring to the amino acid sequence, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “isolated polypeptide” or “isolated peptide” is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide or isolated peptidecontains the polypeptide or peptide in a highly purified form, i.e., atleast 80% pure, at least 90% pure, at least 95% pure, greater than 95%pure, or greater than 99% pure. One way to show that a particularprotein preparation contains an isolated polypeptide or peptide is bythe appearance of a single band following sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis of the protein preparation andCoomassie Brilliant Blue staining of the gel. However, the term“isolated” does not exclude the presence of the same polypeptide orpeptide in alternative physical forms, such as dimers or alternativelyglycosylated or derivatized forms. As was described above, the term “atleast 80% pure” is inclusive of all whole or partial numbers from 80%purity to 100% purity. This same applies to “at least 90% pure” and “atleast 95% pure.” The term “greater than 95% pure” means 95.01% to 100%purity, as described above, and including all whole and partial numbersthere between.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides or peptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or peptide to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a polypeptide or peptide is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete polypeptide orpeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A “detectable label” is a molecule or atom which can be conjugated,attached to or incorporated into a polypeptide to produce a moleculeuseful for diagnosis. The label can be any type of label which, whenattached to or incorporated into a polypeptide renders the polypeptidedetectable. A detectable label may have one or more of the followingcharacteristics: fluorescence, color, radiosensitivity, orphotosensitivity. Examples of detectable labels include chelators,photoactive agents, radioisotopes, fluorescent agents, paramagneticions, or other marker moieties such as a fluorescent resonance energytransfer (FRET) donor and/or acceptor.

The term “affinity tag” is used herein to denote a polypeptide orpeptide segment that can be attached to a second polypeptide or peptideto provide for purification or detection of the second polypeptide orpeptide or provide sites for attachment of the second polypeptide orpeptide to a substrate. In principal, any polypeptide or peptide forwhich an antibody or other specific binding agent is available can beused as an affinity tag. Affinity tags include a poly-histidine tract,protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith andJohnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al.,Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide(Hopp et al., Biotechnology 6:1204 (1988)), streptavidin bindingpeptide, or other antigenic epitope or binding domain. See, in general,Ford et al., Protein Expression and Purification 2:95 (1991). DNAmolecules encoding affinity tags are available from commercial suppliers(e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “substantially similar” when used to describe polypeptide orpeptide sequences or polynucleotide sequences herein means that the twosequences share at least 70% or 75% identity over a corresponding range.More preferably, that percent identity is at least 80% identity, morepreferably still at least 85%, more preferably still at least 90%identity, more preferably still at least 95% identity and mostpreferably at least 96%, 97%, 98% or 99% identity. Differences inidentity can be due to additions, deletions or substitutions of residuesin a first sequences compared to a second sequences. Those ordinarilyskilled in the art will readily calculate percent identity between apolypeptide or peptide sequence or a polynucleotide sequences and areference sequence. For example, the % identity of two polynucleotidesequences may be determined by comparing sequence information using theGAP computer program, version 6.0 described by Devereux et al. (Nucl.Acids Res. 12:387, 1984) and available from the University of WisconsinGenetics Computer Group (UWGCG). Typical default parameters for the GAPprogram include: (1) a unary comparison matrix (comprising a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Various programs known to persons skilled in the art of sequencecomparison can be alternatively utilized.

As is used herein, the terms “at least 70% identical” or “at least 70%identity” means that a polypeptide or peptide sequence or apolynucleotide sequence shares 70%-100% sequence identity with areference sequence. This range of identity is inclusive of all wholenumbers (e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or partial numbers (e.g.,72.15, 87.27%, 92.83%, 98.11%—to two significant figures) embracedwithin the recited range numbers, therefore forming a part of thisdescription. For example, an amino acid sequence with 200 residues thatshare 85% identity with a reference sequence would have 170 identicalresidues and 30 non-identical residues. Similarly, an amino acidsequence with 235 residues may have 200 residues that are identical to areference sequence, thus the amino acid sequence will be 85.11%identical to the reference sequence. Similarly, the terms “at least80%,” “at least 90%,” “at least 95%” and “at least 99%” and the like areinclusive of all whole or partial numbers within the recited range. Asis used herein, the terms “greater than 95% identical” or “greater than95% identity” means that a sequence shares 95.01%-100% sequence identitywith a reference sequence. This range is all inclusive. Differences inidentity can be due to additions, deletions or substitutions of residuesin a first sequences compared to a second sequence.

The term “sample” as used herein includes a biological fluid such asblood, plasma or tissue of a subject. The sample may be obtained orobtainable from a human—such as a human subject—suspected of having adisorder associated with ADAMTS13.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment relates to an ADAMTS13 polypeptide substrate. Suitably,the ADAMTS13 polypeptide substrate is from 45 to 75 amino acids inlength—such as from 45 to 72 amino acids in length or from 45 to 70amino acids in length or from 50 to 75 amino acids in length. Moresuitably, the ADAMTS13 polypeptide substrate is from 45 to 65 aminoacids in length, from 50 to 65 amino acids in length, from 50 to 60amino acids in length, from 51 to 59 amino acids in length, from 52 to58 amino acids in length, from 53 to 57 amino acids in length, from 54to 56 amino acids in length, from 50 to 70 amino acids in length, from55 to 70 amino acids in length, from 55 to 65 amino acids in length,from 60 to 65 amino acids in length, from 61 to 64 amino acids in lengthor from 61 to 63 amino acids in length. In one embodiment, the ADAMTS13polypeptide substrate is from 55 to 62 amino acids in length. In oneembodiment, the ADAMTS13 polypeptide substrate is 55 amino acids inlength. In one embodiment, the ADAMTS13 polypeptide substrate is 62amino acids in length. In one embodiment, the ADAMTS13 polypeptidesubstrate is 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 aminoacids in length and contains a feature as described herein.

The ADAMTS13 polypeptide substrate is an isolated chimeric or mutantamino acid construct encompassing portions of the VWF A2 domain.

In one aspect, the isolated polypeptide substrate is from 45 to 70 aminoacids in length and has an amino acid sequence that is substantiallysimilar to part of the VWF A2 domain sequence set forth in SEQ ID NO: 2,with one or more of the following modifications: (i) the amino acidcorresponding to position 1599 of SEQ ID NO: 2 is mutated from Q to K;(ii) the amino acid corresponding to position 1610 of SEQ ID NO: 2 ismutated from N to C; and (iii) the amino acids corresponding to Q1624 to11642 of SEQ ID NO: 2 are deleted.

In another aspect, the isolated polypeptide substrate is from 50 to 75amino acids in length and has an amino acid sequence that issubstantially similar to part of the VWF A2 domain sequence set forth inSEQ ID NO: 2, with one or more of the following modifications: (i) theamino acid corresponding to position 1599 of SEQ ID NO: 2 is mutatedfrom Q to K; and (ii) the amino acid corresponding to position 1610 ofSEQ ID NO: 2 is mutated from N to C; and (iii) the amino acidcorresponding to position 1629 of SEQ ID NO: 2 is mutated from G to E;and (iv) the amino acids corresponding to G1631 to R1641 of SEQ ID NO: 2are deleted.

Suitably, the amino acid at the N-terminus of said polypeptide substratecorresponds to D1596 of SEQ ID NO: 2. Suitably, the amino acid at theC-terminus of said polypeptide substrate corresponds to R1668 of SEQ IDNO: 2. Suitably, the amino acid at the N-terminus of said polypeptidesubstrate corresponds to D1596 of SEQ ID NO: 2 and the amino acid at theC-terminus of said polypeptide substrate corresponds to R1668 of SEQ IDNO: 2.

SEQ ID NO: 2 corresponds to a fragment of the A2 domain of VWF from HomoSapiens; Accession number P04275-1 (UniProtKB/Swiss-Pro); UPI0001BBE42F(UniParc); IPI00023 014.2 (International Protein Index).

In one embodiment, the ADAMTS13 polypeptide substrate comprises,consists or consists essentially of the sequence set forth in SEQ ID NO:7 or SEQ ID NO: 1 or a sequence that has substantial identity thereto.Isomers thereof are also contemplated. According to a furtherembodiment, the ADAMTS13 polypeptide substrate may comprise one or morefurther amino acids at the N-terminus or the C-terminus or theN-terminus and the C-terminus of the polypeptide substrate.

Cleavage products of the SEQ ID NO: 1 or SEQ ID NO: 7 polypeptidesubstrate are also disclosed, particularly those cleavage productsgenerated following fragmentation with ADAMTS13. In particular,C-terminal fragments are disclosed. Thus, in a further aspect there isprovided an isolated polypeptide substrate for a disintegrin-like andmetallopeptidase with thrombospondin type-1 motif, 13 (ADAMTS13) that isor is at least 52 (fifty two) amino acids in length and has an aminoacid sequence that is substantially similar to part of the vonWillebrand factor A2 domain sequence set forth in SEQ ID NO: 2, with oneor more of the following modifications: (i) the amino acid correspondingto position 1610 of SEQ ID NO: 2 is mutated from N to C; and (ii) theamino acids corresponding to Q1624 to R1641 of SEQ ID NO: 2 are deleted.Suitably, the amino acid at the N-terminus of said polypeptide substratecorresponds to M1606 of SEQ ID NO: 2. Suitably, the amino acid at theC-terminus of said polypeptide substrate corresponds to R1668 of SEQ IDNO: 2. Suitably, said polypeptide is a synthetic polypeptide thatcomprises at least one portion of a detectable label. Suitably, at leastone portion of the detectable label is a fluorophore or a quencher.Suitably, the attachment site for the fluorophore or the quencher is atthe amino acid corresponding to position 1610 of SEQ ID NO: 2. Inanother aspect, there is provided an isolated polypeptide substrate fora disintegrin-like and metallopeptidase with thrombospondin type-1motif, 13 (ADAMTS13) that is or is at least 45 (forty five) amino acidsin length and has an amino acid sequence that is substantially similarto part of the von Willebrand factor A2 domain sequence set forth in SEQID NO: 2, with one or more of the following modifications: (i) the aminoacid corresponding to position 1610 of SEQ ID NO: 2 is mutated from N toC; (ii) the amino acid corresponding to position 1629 of SEQ ID NO: 2 ismutated from G to E; and (iii) the amino acids corresponding to G1631 toR1641 of SEQ ID NO: 2 are deleted. Suitably, the amino acid at theN-terminus of said polypeptide substrate corresponds to M1606 of SEQ IDNO: 2. Suitably, the amino acid at the C-terminus of said polypeptidesubstrate corresponds to R1668 of SEQ ID NO: 2. Suitably, saidpolypeptide is a synthetic polypeptide that comprises at least oneportion of a detectable label. Suitably, the at least one portion of thedetectable label is a fluorophore or a quencher. Suitably, theattachment site for the fluorophore or the quencher is at the amino acidcorresponding to position 1610 of SEQ ID NO: 2.

Isolated nucleotide sequences encoding the polypeptide substratesdescribed herein are also disclosed. In addition, functional fragmentsof VWF genes are disclosed. Within the context of this disclosure, a“functional fragment” or “fragment” of a VWF gene refers to a nucleicacid molecule that encodes a portion of a VWF polypeptide which is adomain described herein or at least specifically interacts with ADAMTS13as a substrate for the cleavage activity of ADAMTS13. A functionalfragment of the VWF gene need not encode a polypeptide that containseach contiguous amino acid residue of the portion of VWF to which thefunctional fragment corresponds. In other words, the function fragmentof VWF can align to a portion of native VWF and can include one or moreof an insertion, a deletion or a substitution, so long as the functionalfragment is a substrate to ADAMTS13 cleavage activity.

VWF is a large multimeric plasma glycoprotein crucial in the maintenanceof hemostasis by functioning as both an antihemophilic factor carrierand a platelet-vessel wall mediator in the blood coagulation system,mainly by mediating tethering and adhesion of circulating platelets atsites of vascular injury. Mutations in this gene or deficiencies in thisprotein result in von Willebrand's disease (VWD).

VWF is expressed by endothelial cells and megakaryocytes. It issynthesized as 250-kDa monomers, which undergo intracellular processing,glycosylation, multimerization and propeptide removal that leads toformation of mature VWF multimers.

VWF multimeric size is modulated by the plasma metallopeptidase ADAMTS13(a disintegrin and metallopeptidase with thrombospondin type I motif,member 13), which cleaves at a single site in the VWF A 2 domain(AA1498-1665; UniProtKB/Swiss-Pro database; Accession: P04275; FIG. 1)between Y1605 and M1606 (FIG. 2).

As described herein, a synthetic 55 (fifty five) amino acids (AA) inlength polypeptide sequence designated as “GTI_FRETS” SEQ ID NO: 7 isdisclosed, optionally modified with the insertion of a detectablelabel—such as a quencher and a fluorophore, that when recognized andcleaved by ADAMTS13 emits fluorescence. A synthetic 62 (sixty two) aminoacids (AA) in length polypeptide sequence designated as “GTI_FRET4” SEQID NO: 1 is also disclosed, optionally modified with the insertion of adetectable label—such as a quencher and a fluorophore, that whenrecognized and cleaved by ADAMTS13 emits fluorescence.

Suitably, the polypeptide(s) are prepared using chemical synthesistechniques that are known in the art. The synthesis may utilize solid-or liquid-phase peptide synthesis. When modification of amino acidresidues is required, modified amino acids can be introduced into apeptide synthesizer as appropriate.

It is also possible to produce the polypeptide substrates by recombinantprocedures. Production of polypeptides by recombinant procedures can becarried out by methods well known to those skilled in the art, such asmethods described by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Suitably, the polypeptide(s) can be lyophilized polypeptide(s).Lyophilization can be carried out according to procedures known to thoseskilled in the art, such as methods described in U.S. Pat. No. 5,556,771and references therein.

The activity of ADAMTS13 in a subject can be measured using thepolypeptide substrate for ADAMTS13. For example, the polypeptidesubstrate can be contacted with a sample from a subject—such asplasma—and the resultant polypeptide fragments of the polypeptidesubstrate are analysed. Various methods in the art can be used toanalyse the resultant polypeptide fragments including the use ofSDS-polyacrylamide gel electrophoresis. The proteins are stained using,for example, Coomassie Blue or silver staining or the like and thefragments produced are analysed. Alternatively, it may be possible tocarry out Western blotting following the SDS-PAGE. Suitably, the resultsare compared with a control sample and/or a calibrator sample. Thecontrol sample may be or may be derived from a subject who is known tohave ‘normal’ activity of ADAMTS13, such that a diagnosis of abnormalactivity can be made.

Although the detectable label may be directly attached to an amino acidresidue of a polypeptide, a detectable label may also be indirectlyattached, for example, by being complexed with a chelating group that isattached (for example, linked via a covalent bond or indirectly linked)to an amino acid residue of the polypeptide. In a particular embodiment,the “detectable label” is any type of label that only substantiallyreleases a detectable signal once the polypeptide substrate is cleaved.Thus, the detectable label may comprise a fluorescent resonance energytransfer (FRET) donor and/or acceptor. In one embodiment, thepolypeptide substrate is modified by the incorporation or insertion ofat least one quencher and at least one fluorophore, so that whenrecognized and cleaved by ADAMTS13 emits fluorescence. Suitably thesubstrate is a synthetic polypeptide (in contrast to a recombinantpolypeptide) since this allows the direct incorporation of a quencher(s)and a fluorophore(s) therein. In the uncleaved substrate, fluorescenceresonance energy transfer between the quencher and the fluorophore leadsto low (for example, substantially no) fluorescence. Upon cleavage ofthe substrate by ADAMTS13, the quencher and fluorophore are separatedwhich results in a detectable increase in fluorescence which can bemeasured.

Thus, in one embodiment, the polypeptide substrate includes a detectablelabel that allows the fragmentation of the polypeptide substrate to bemeasured directly. In one particular embodiment, the detectable label isa fluorophore and a quencher, wherein the quenching of the fluorophoreis diminished as fragmentation occurs. Accordingly, fragmentation of theADAMTS13 polypeptide substrate results in an increase in fluorescentsignal. The cleavage of the substrate is detected by reading thefluorescence that results when the substrate is cleaved. According tothe this embodiment of the invention, the skilled person will recognizethat the polypeptide substrate will need to be synthesised by chemicalsynthesis techniques since recombinant approaches do not typically allowthe incorporation of detectable labels therein.

The attachment site for the fluorophore and the quencher will typicallybe within the polypeptide substrate. Suitably, the fluorophore and thequencher will be separated from each other in such a manner thatfluorescence from the fluorophore is substantially quenched when thepolypeptide substrate is intact and fluorescence from the fluorophore isnot quenched once the polypeptide substrate is cleaved. In oneembodiment, the fluorophore and the quencher are separated by 8, 9, 10,11 or 12 amino acids, suitably, the fluorophore and the quencher areseparated by 9, 10, or 11 amino acids, more suitably, the fluorophoreand the quencher are separated by 10 amino acids. In one embodiment, theattachment site for the fluorophore is at the amino acid correspondingto position 1610 of SEQ ID NO: 2 and/or the attachment site for thequencher is at the amino acid corresponding to position 1599 of SEQ IDNO: 2. It also contemplated that the positions of the fluorophore andquencher are reversed such that the quencher is at the amino acidcorresponding to position 1610 of SEQ ID NO: 2 and/or the attachmentsite for the fluorophore is at the amino acid corresponding to position1599 of SEQ ID NO: 2.

Another aspect relates to a method for measuring the activity ofADAMTS13 in a sample, which comprises contacting the polypeptidesubstrate described herein with a sample from a subject and analyzingthe fragmentation products thereof.

There is also disclosed a kit or a diagnostic composition for in vitrotesting of the ADAMTS13 activity in a subject (for example, a decreaseor deficiency of ADAMTS13 activity) and therefore the presence of TTP orthe predisposition to TTP, or for making a definitive diagnosis of TTPand a discrimination between TTP and HUS. Mild or moderately decreasedlevels of ADAMTS13 activity have also been associated with other diseasestates and conditions (see, for example, Kokame et al. Blood (2004) 103,607; and Kokame et al. Br. J. Haematol (2005) 129, 93). The kit or thecomposition comprises a polypeptide substrate for ADAMTS13 as describedherein. Typically, the kit will also include a one or more positivecontrols and/or one or more calibrators. Typically, the kit will alsoinclude a specimen diluent and/or a substrate buffer (for example, abuffer solution whose pH corresponds to a pH range of 5.8 to 6.7 that issuitable for in vitro testing of the proposed polypeptide substrates.).A set of instructions may also be provided. Methods for carrying out thein vitro testing of the ADAMTSI3 activity in a subject are known in theart (see e.g., Miyata, T., K. Kokame, F. Banno, Y. Shin, and M. Akiyama.2007. ADAMTSI3 assays and ADAMTS13-deficient mice. Curr. Opin. Hematol.14:277-283). Numerous vendors sell kits for detecting and/or determiningthe activity of ADAMTS-13 (see e.g., FRETS-VWF73 (PeptidesInternational, U.S.A., Cat# SFR-3224-s), TECHNOZYM® ADAMTS-13 INH ELISA(Kordia, Netherlands, Cat# TC 5450401), Human ADAMTS13 ELISA Kit andADAMTS13 Antibody Agarose Immobilized (both available from BethylLaboratories, U.S.A., Cat#s E88-500 and S300-391) and IMUBIND® ADAMTS13ELISA (American Diagnostica, GmbH, Germany, Cat#813). Methods forcollecting, transporting and processing blood specimens for coagulationtesting and general performance of coagulation assays are known in theart (see for example, Approved Guideline H21-A4 NCCLS, Volume 23, Number35, December 2003; Br. J. Haematol. 129:93-100 and Proc Natl Acad SciUSA. 2006; 103: 18470-4). The kit can also include an activator ofADAMTS13-such as divalent metal ions.

The polypeptide substrate may have a tag sequence attached at theN-terminus and/or at the C-terminus thereof. The tag sequence may beuseful in the detection, quantification, or separation of cleavedproducts. Also, the tag sequence may be useful for immobilizing thepolypeptide substrate onto a solid phase. Thus, the present inventionalso encompasses polypeptide substrates which are immobilized onto asolid phase using such tag sequences. The tag sequence can include, butare not limited to, proteins (for example, glutathione transferase,luciferase, beta-galactosidase), peptides (for example, His tags),coupling agents (for example, carbodiimide reagents), various kinds oflabels (for example, radioactive labels, chromophores, and enzymes).

In further embodiments, the present invention relates to use of thepolypeptide substrate for producing the diagnostic composition or thekit as described above.

The disclosure is further described in the Examples below, which areprovided to describe the invention in further detail. These examples,which set forth a preferred mode presently contemplated for carrying outthe invention, are intended to illustrate and not to limit theinvention.

EXAMPLES Example 1: Evaluation of SEQ ID NO: 1 Polypeptide Substrate(GTI_FRET4) as an ADAMTS13 Substrate

Purpose:

The purpose of this experiment was to evaluate the polypeptide of SEQ IDNO: 1 (GTI FRET4; 62AA; MW 7855.9; polypeptide purity 95.5%) for use asan ADAMTS13 substrate in a second generation ADAMTS13 assay.

Synopsis of the Procedure:

The procedure in this example was performed substantially as describedin Kokame, K., Y. Nobe, Y. Kokubo, A. Okayama, and T. Miyata. 2005.FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br. J.Haematol. 129:93-100, but using SEQ ID NO: 1 in place of the 73 aminoacid substrate described therein. The FRETS-VWF73 substrate solution wasdissolved in 25% dimethyl sulphoxide/water to prepare the 100 microMstock solution. The GTI FRET4 SEQ ID NO: 1 was dissolved in 100% DMSO.Both substrates were diluted to equal concentrations using ATS-13substrate buffer (Gen-Probe GT1 Diagnostics, Inc., U.S.A., Cat# ATS-13).

Plasma samples were diluted according to the ATS-13 Direction Insertusing ATS-13 specimen diluent (Gen-Probe GTI Diagnostics, Inc., U.S.A.,Cat# ATS-13). The diluted plasma samples were mixed with the dilutedsubstrate and the fluorescence was read at 0, 5, 10, 15, 20, 30, 45minutes using a Biotek FLX800 at the appropriate excitation and emissionwavelengths for each substrate. The fluorescence values are reported inTable 1.

The fluorophore-quencher pair in the SEQ ID NO: 1 polypeptide substrateis FAM-5/TQ 2™ (Ex 485±20; Em 528±20; AAT Bioquest, Inc. Sunnyvale,Calif. U.S.A.). The fluorophore and quencher pair of FRETS-VWF73(Nma/Dnp) described in Kokame has been substituted with FAM-5 and TQ_2respectively in GTI_FRET4 SEQ ID NO: 1. Furthermore, in the SEQ ID NO: 1polypeptide the position of the fluorophore (FAM-5) and quencher (TQ_2)has been swapped relative to the position of the fluorophore andquencher of the FRETS-VWF73 construct. Therefore, for GTI_FRET4 SEQ IDNO: 1, attachment of the fluorophore (FAM-5) occurs by substitutingasparagine with cystine at position 15. The quencher (TQ_2) was attachedby substituting glutamine with lysine at position 4 (FIG. 2 and Table4).

Results:

The results of this experiment demonstrate that by using SEQ ID NO: 1polypeptide substrate, as compared to the prior art FRETS-VWF73substrate, a larger dynamic range is obtained. In this experiment, at 30minutes there was approximately 34,000 Relative Fluorescence Units (RFU)difference between Calibrator A (equivalent to 0% of ADAMTS13 activity)and Calibrator E (equivalent to 100% of ADAMTS13 activity) compared toapproximately 1500 to 2000 RFU difference for the FRETS-VWF73 substrate.See Table 1 and FIG. 3 and FIG. 4 for the change in fluorescenceobserved at all time points.

The calibration curves result in a linear trend line (see FIG. 4)compared to the FRETS-VWF73 assay which produces a calibration curverequiring a polynomial trend line (see FIG. 3). The calibration curvefor the SEQ ID NO: 1 polypeptide substrate continued to be linear upthrough 45 minutes.

The % Normal (% N) activity (see Table 2) is calculated using the lineartrend lines observed from the calibration curve at each time point. The% N ADAMTS13 activity calculated for each sample plateaus at 30 minutesand shows comparable results to FRETS-VWF73 after only 15 minutes.

Example 2: Direct Comparison of SEQ ID NO: 1 Polypeptide Substrate(GTI_FRET4) and the Prior Art FRETS-VWF73 Polypeptide Substrate(Peptides International; Louisville, Ky.)

Purpose:

The purpose of this experiment is to compare the SEQ ID NO: 1polypeptide substrate to FRETS-VWF73 (SEQ ID NO: 6).

Synopsis of the Procedure:

For this experiment, substrate concentration and fluorescence readersettings determined on the previous experiment are used. The specimenstested (listed in Table 3), include a panel of proficiency samplesprepared for use with ATS-13 (Gen-Probe GTI Diagnostics, Inc., U.S.A.,Cat# ATS-13) which included samples with normal or deficient ADAMTS13activity levels. In addition, six Factor Assay ConTrol plasma were used(2 FACT, 2 A-FACT and 2 B-FACT, from George King Biomedical Inc.,Kansas, USA). The assays for the SEQ ID NO: 1 substrate and for theprior art FRETS-VWF73 assay were performed generally as according to theprocedure described in Example 1. Substrate is prepared according to theconditions used for initial testing of the substrate, which prepared themolar amount of SEQ ID NO: 1 polypeptide substrate used in the assay tobe equivalent to the molar amount of FRETS-VWF73 used in the prior art.The ELISA assays were read at 0, 5, 10, 15, 20, 30, 45 minutes.

Results

The results of these experiments confirm that the SEQ ID NO: 1polypeptide substrate provides a much larger dynamic range compared tothe FRETS-VWF73 substrate. At 30 minutes the difference betweenCalibrator A and E is approximately 35000 RFU compared to 2500 RFUobserved for FRETS-VWF73 (FIGS. 3-4). The larger dynamic range wouldresult in better sensitivity for samples with low ADAMTS13 activity.Moreover, when used at the same concentration as the FRETS-VWF73substrate, the reaction time is faster. Consistent % N activity valuesare observed by the 15 minute reading (Table 2). The calibration curvesare linear which would eliminate complicated analysis of results for theuser.

Example 3: Evaluation of the Cleavage of SEQ ID NO: 7 PolypeptideSubstrate (GTI_FRETS)

The purpose of this experiment is to compare the SEQ ID NO: 7polypeptide substrate with the SEQ ID NO: 1 polypeptide substrate.

Synopsis of the Procedure:

Testing of substrate for cleavability by ADAMTS13 is determined as isgenerally described in Example 1.

Results:

The change in fluorescence with time is shown in FIG. 5 and demonstratesthat cleavage of the SEQ ID NO: 7 polypeptide substrate occurs.

Example 4: Evaluation of the Solubility of the SEQ ID NO: 7 PolypeptideSubstrate and Assay Analysis

The purpose of this experiment is to evaluate the solubility of the SEQID NO: 7 polypeptide substrate and to compare its performance with theSEQ ID NO: 1 polypeptide substrate.

250 .micro.L of working solution is prepared as above. The solution isvortexed vigorously and appears to be in solution. The solution iscentrifuged at ˜12,000 g for about 2 minutes. After centrifugation, avery small pink pellet is noted at the bottom of the tube. This suggeststhat at least some amount of the material precipitates. Another tube isprepared as above substituting water for the substrate buffer. This tubeis also centrifuged. Once again a pink pellet is observed in the bottomof the tube. The pellet observed in the water solution is noticeablylarger than the pellet observed in the substrate buffer solution. Thissuggests that the polypeptide is less soluble in water than in thebuffer.

The purpose of this experiment is to compare the SEQ ID NO: 7 GTI_FRETSpolypeptide substrate to GTI_FRET4 (SEQ ID NO: 1). Testing of substratefor cleavability by ADAMTS13 is determined as is generally described inExample 1 but using GTI_FRETS instead of FRETS-VWF73.

The assay is read at 0, 5, 10, 15, 20, 30, 45, 60, and 90 minutes.

Results:

The data from this experiment demonstrates that the SEQ ID NO: 7polypeptide substrate is not completely soluble in the working solutionas prepared. However, the resulting calibration curve that is obtainedis linear (see FIG. 5) and cleavage of the substrate occurs. Acomparison of the activity obtained using SEQ ID NO: 1 polypeptidesubstrate or SEQ ID NO: 7 polypeptide substrate at 30 minutes postaddition of substrate is shown in Table 5. The calibration curve for theSEQ ID NO: 7 polypeptide substrate continued to be linear up through 60minutes.

Any publication cited or described herein provides relevant informationdisclosed prior to the filing date of the present application.Statements herein are not to be construed as an admission that theinventors are not entitled to antedate such disclosures. Allpublications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedisclosure will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled the art are intended to bewithin the scope of the following claims.

TABLE 1 Change in fluorescence observed using the SEQ ID NO: 1 peptidesubstrate Increase in Fluorescence Values at Each Time Point (X minute-0minute Reading) 15 30 45 60 75 90 Sample ID min min min min min minCalibrator A 676 843 921 1,166 1,013 959 Calibrator B 1,765 3,000 4,1645,358 6,468 7,581 Calibrator C 6,178 11,701 16,807 21,705 26,685 32,209Calibrator D 10,887 20,626 29,206 37,289 44,543 53,613 Calibrator E18,718 35,085 49,418 61,068 70,603 82,999 Positive 9,146 17,281 24,85931,857 39,838 47,473 Control High Positive 2,182 3,902 5,511 7,169 8,78910,370 Control Low VF 1,292 2,436 3,552 4,690 5,784 7,260 UAMS0416093,876 7,931 11,692 15,886 19,867 24,709 MON110707 17,881 33,432 46,84858,747 68,370 78,371

TABLE 2 Calculated % N activity using the linear trend line obtained foreach time point using the SEQ ID NO: 1 peptide substrateAssigned/Expected % Normal ADAMTS13 Activity Values Based on FRETS-VWF73% Normal ADAMTS13 Values Calculated Substrate Using GTI_FRET4 SubstrateIncubation Time 30 15 30 45 60 75 90 Sample ID Minutes Minutes MinutesMinutes Minutes Minutes Minutes Calibrator A 0 1 1 0 0 0 0 Calibrator B9 7 7 7 7 6 6 Calibrator C 34 32 33 33 34 35 36 Calibrator D 55 58 59 5960 61 62 Calibrator E 102 101 101 100 100 98 98 Positive 36-54 48 49 5051 54 55 Control High Positive  6-17 10 10 10 10 10 9 Control Low VF ~205 6 6 6 5 6 UAMS041609 ~35 19 22 23 24 26 27 MON110707 ~90-100 97 96 9596 95 92

TABLE 3 Materials used performing the examples Manufacturer (Cat. No.)Material or Associated Date Lot No. ATS-13 GTI CA-CE020410,Calibrators/Controls PCH020410, PCL020410 Substrate buffer (SBA) GTISBA011810 Specimen diluent (SDA) GTI SDA011810 Substrate (SA) GTISA112509 Plate GTI ATS-011410 DMSO Sigma (D8418) 038K07101 Normal PooledPlasma GTI NPP032206 ATS-13 Proficiency GTI 020910-ATS Samples 1-5A-FACT plasma George King BioMedical 1284 (A-FACT) A-FACT plasma GeorgeKing BioMedical  900 (A-FACT) B-FACT plasma George King BioMedical 1114(B-FACT) B-FACT plasma George King BioMedical 1266 (B-FACT) FACT plasmaGeorge King BioMedical 1223 (FACT) FACT plasma George King BioMedical 222e1 (FACT) VF Apr. 9, 2001 Mar. 8, 2010 BCM2 Jul. 22, 2008 Jul. 22,2008 UAMS041609 Apr. 16, 2009 Apr. 16, 2009 ATS AC and AB CNTL 05/14/2092051008

TABLE 4 Amino acid sequences SEQ ID NO:Sequence. N-terminus to C-Terminus. note 1DREKAPNLVYMVTGCPASDEIKRLPGDIQVVPIEVIGWPNAPILIQDFETLP GTI FRET4REAPDLVLQR 2 MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYS1-22 Signal FAGYCSYLLAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQPeptide; GDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFN 23-763vonKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSS willebrandSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGG antigen II;LECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTC 764-2813QSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLS vwf.RDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNOGDDFLTPSGLAEPRVEDFGNAWKLHGDCODLOKOESDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGETVKIGCNTCVCODRKWNCTDHVCDATCSTIGMAHYLTEDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNEDGIQNNDLTSSNLQVEEDPVDEGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVELLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEEMEEVIORMDVGODSIHVTVLOYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSP RKCSK 3MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSP ADAMTS13GAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYV Isoform 1.LTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCG 1-29 signalWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSC Peptide;LITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGL 30-74AWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCR propeptide;VAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEK 75-1427WCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRP ADAMTS13AFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFY chain.HWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQOPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVOLDOGODVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPAPQPRRLLPGPQENSVQSSACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTLQSWVPEMQDPQSWKGKEGT 4MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSP ADAMTS13GAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYV Isoform 2.LTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPDAQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQOPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVOLDOGODVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTL QSWVPEMQDPQSWKGKEGT5 MHQRHPRARCPPLCVAGILACGFLLGCWGPSHFQQSCLQALEPQAVSSYLSP ADAMTS13GAPLKGRPPSPGFQRQRQRQRRAAGGILHLELLVAVGPDVFQAHQEDTERYV Isoform 3.LTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAAPRAGLAWSPCSRRQLLSLLSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPLDQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCGGGVVTRRRQCNNPRPAFGGRACVGADLQAEMCNTQACEKTQLEFMSQQCARTDGQPLRSSPGGASFYHWGAAVPHSQGDALCRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGSCRTFGCDGRMDSQQVWDRCQVCGGDNSTCSPRKGSFTAGRAREYVTFLTVTPNLTSVYIANHRPLFTHLAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVEYRVALTEDRLPRLEEIRIWGPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQAWVWAAVRGPCSVSCGAGLRWVNYSCLDQARKELVETVQCQGSQQPPAWPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKTLPPARCRAGAQOPAVALETCNPOPCPARWEVSEPSSCTSAGGAGLALENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDATSAGEKAPSPWGSIRTGAQAAHVWTPAAGSCSVSCGRGLMELRFLCMDSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCGRGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEAACAALVRPEASVPCLIADCTYRWHVGTWMECSVSCGDGIQRRRDTCLGPQAQAPVPADFCQHLPKPVTVRGCWAGPCVGQGACGRQHLEPTGTIDMRGPGQADCAVAIGRPLGEVVTLRVLESSLNCSAGDMLLLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWGEIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANASYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQASLRGQYWTLQSWVPEMQDPQSWKGKEGT 6DREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVOELERIGWPNA FRETS-VWF73PILIQDFETLPREAPDLVLQR 7DREKAPNLVYMVTGCPASDEIKRLPGDIIGWPNAPILIQDFETLPREAPDLV GTI FRETS LQR

TABLE 5 A comparison of the activity obtained using SEQ ID NO: 1polypeptide substrate versus SEQ ID NO: 7 polypeptide substrate at 30minutes post addition of substrate % Normal ADAMTS13 Activity: 30 MinuteIncubation Time Sample ID GTI_FRET5 Substrate GTI_FRET4 SubstrateCalibrator A  3 4 Calibrator B  8 9 Calibrator C 30 29 Calibrator D 6461 Calibrator E 108  110 Positive Control High 46 50 Positive ControlLow 13 14 90 (ATS13-1) 65 92 72 (ATS13-2) 52 72 50 (ATS13-3) 40 47 22(ATS13-4) 18 21 5 (ATS13-5)  6 7 UAMS041609 14 31 BCM2 22 19 VF040901 1010 CNTL <Calibrator A <Calibrator A NPP032206 84 >Calibrator E NPP032206HI 12 9 NPP032206 mixed <Calibrator A 57 BCM2 HI 15 12 BCM2 mixed 37 51CNTL HI <Calibrator A <Calibrator A CNTL mixed <Calibrator A 4 NPP032206at 37 C. 77 106 A-FACT lot 1284 <Calibrator A 10 A-FACT lot 900<Calibrator A 10 B-FACT lot 1114 28 45 B-FACT lot 1266 23 46 FACT lot1223 82 107 FACT lot 222e1 85 111

The invention claimed is:
 1. An isolated polypeptide substrate for adisintegrin-like and metallopeptidase with thrombospondin type-1 motif,13 (ADAMTS13), wherein said polypeptide substrate is from 45 to 70 aminoacids in length and comprises: (a) the amino acid sequence set forth inSEQ ID NO:7; or (b) an amino acid sequence that is substantially similarto SEQ ID NO:7, wherein: (i) the amino acid corresponding to position 4of SEQ ID NO:7 is lysine (K); and (ii) the amino acid corresponding toposition 15 of SEQ ID NO:7 is cysteine (C).
 2. The polypeptide substrateof claim 1 comprising the amino acid sequence set forth in SEQ ID NO:7.3. The polypeptide substrate of claim 1, wherein said polypeptidesubstrate comprises a detectable label.
 4. A kit comprising apolypeptide substrate of claim 1 and one or more calibrators containinga known concentration of ADAMTS13 activity and/or one or more positivecontrols for ADAMTS13 activity.
 5. An isolated polypeptide substrate fora disintegrin-like and metallopeptidase with thrombospondin type-1motif, 13 (ADAMTS13), wherein said polypeptide substrate is from 50 to75 amino acids in length and comprises: (a) the amino acid sequence setforth in SEQ ID NO:1; or (b) an amino acid sequence that issubstantially similar to SEQ ID NO:1, wherein: (i) the amino acidcorresponding to position 4 of SEQ ID NO:1 is lysine (K); (ii) the aminoacid corresponding to position 15 of SEQ ID NO:1 is cysteine (C); and(iii) the amino acid corresponding to position 34 of SEQ ID NO:1 isglutamic acid (E).
 6. The polypeptide substrate of claim 5, wherein theamino acid at the N-terminus of said polypeptide substrate is asparticacid (D).
 7. The polypeptide substrate of claim 5, wherein the aminoacid at the C-terminus of said polypeptide substrate is arginine (R). 8.The polypeptide substrate of claim 5, comprising the amino acid sequenceset forth in SEQ ID NO:1.
 9. The polypeptide substrate of claim 5,wherein said polypeptide substrate comprises a detectable label.
 10. Thepolypeptide substrate of claim 9, wherein said detectable labelcomprises a fluorophore and a quencher.
 11. The polypeptide substrate ofclaim 10, wherein: (i) said fluorophore is attached to cysteine (C)corresponding to position 15 of SEQ ID NO:1; and said quencher isattached to lysine (K) corresponding to position 4 of SEQ ID NO:1; or(ii) said quencher is attached to cysteine (C) corresponding to position15 of SEQ ID NO:1; and said fluorophore is attached to lysine (K)corresponding position 4 of SEQ ID NO:1.
 12. A method for measuringADAMTS13 activity in a sample comprising: contacting said sample with apolypeptide substrate of claim
 5. 13. The method of claim 12 whereinsaid measuring ADAMTS13 activity comprises detecting cleavage of saidpolypeptide substrate.
 14. The method of claim 12, wherein saidpolypeptide substrate is attached to a solid support.
 15. The method ofclaim 12, wherein said sample is a biological fluid or a tissue sample.16. The method of claim 15, wherein said biological fluid is a bloodsample or a plasma sample.
 17. The method of claim 12, wherein saidsample is obtained from a human subject.
 18. A kit comprising apolypeptide substrate of claim 5 and one or more calibrators containinga known concentration of ADAMTS13 activity and/or one or more positivecontrols for ADAMTS13 activity.
 19. The kit of claim 18 furthercomprising at least one of: a specimen diluent, a substrate buffer, andinstructions for using said kit.
 20. A method for cleaving a polypeptidecomprising: contacting said polypeptide substrate of claim 5 with anADAMTS13 protease.